l-Chloro-2,4-dinitrobenzene(CDNB), a halogenated nitro derivative of hydrocarbon, is a potent skin sensitizer causing severe allergic contact dermatitis, and thus is a documented occupational hazard as well. Previous studies from this laboratory have shown that CDNB induces microtubule (MT) disassembly in mouse 3T3 cells. Blocking of certain -SH groups of tubulin, the principal protein component of MT, with -SH reagents inhibits tubulin polymerization in vitro. Since glutathione (GSH) is the predominant non-protein thiol in the cell, it is reasonable to expect that GSH is involved in the dynamics of MT state. Furthermore, GSH is crucial for cellular defense against insults by toxic electrophiles by forming appropriate conjugates with the toxicants through the action of glutathione-S-transferase (GST). However, the mechanisms by which CDNB induces cytoskeletal perturbation and its relationship to the ultimate cellular injury are not clear. Therefore, the specific aims of my project are: 1) to determine whether CDNB acts directly on the cytoskeleton by binding to tubulin/MT in the cells resulting in MT disassembly; 2) to investigate the role of total cellular can content, which can be manipulated by the use of specific agents to either increase or decrease, in modulating the dynamics of MT assembly and disassembly and microfilament (MF) distribution during CDNB exposure; and 3) to quantitate the amount of cellular free vs polymerized tubulin and actin (the major protein of MF) by developing an ELISA assay for each protein. Studies on the relationship between the cellular GSH contents and the dynamic changes in the cytoskeleton of human fibroblasts may lead to a better understanding of the mechanisms of induction of allergic contact dermatitis caused by CDNB and other related halogenated nitro derivatives of hydrocarbon.